Methods for increasing stearate content in soybean oil

ABSTRACT

This invention relates to a method for increasing stearate as a component of total triglycerides found in soybean seed. The method generally comprises growing a soybean plant having integrated into its genome a DNA construct comprising, in the 5′ to 3′ direction of transcription, a promoter functional in a soybean plant seed cell, a DNA sequence encoding an acyl-ACP thioesterase protein having substantial activity on C18:0 acyl-ACP substrates, and a transcription termination region functional in a plant cell. The present invention also provides a soybean seed with about 33 weight percent or greater stearate as a component of total fatty acids found in seed triglycerides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/305,021, filed Dec. 19, 2005, which is a continuation of U.S.application Ser. No. 10/061,280, filed Feb. 4, 2002, which is acontinuation of Ser. No. 09/359,070, filed Jul. 22, 1999, now U.S. Pat.No. 6,380,462, which is a continuation-in-part of U.S. application Ser.No. 09/134,262, filed Aug. 14, 1998, now U.S. Pat. No. 6,365,802. Theentirety of these applications are hereby incorporated by reference.

INCORPORATION OF SEQUENCE LISTING

A paper copy of the Sequence Listing is submitted herewithelectronically via EFS web. A computer-readable form of the SequenceListing is also submitted herewith electronically via EFS web andcontains the file named “17030-0004_corr_seqlist.txt”, which is 5,153bytes in size (measured in MS-DOS) and which was created on Apr. 1,2009. The sequence listings submitted herewith as paper copy andcomputer-readable form are herein incorporated by reference.

INTRODUCTION

1. Field of the Invention

The invention relates to genetic modification of plants, plant cells andseeds, particularly altering fatty acid composition.

2. Background

Soybean (Glycine max) is one of the highest value crops currently grownin the United States (≈$16 billion in 1996). Ranking close to corn (25%)and wheat (22%), soybean accounted for 19% of the United States cropacres planted in 1994. Often referred to as a “miracle crop”, soybeanoffers tremendous value through the oil, protein and whole soybeanproducts. Agronomic traits, food quality traits related to oils andprotein quality are all important for the soybean industry.

More soybeans are grown in the United States than anywhere else in theworld (2.4 billion bushels in 1996, 50% of world production). A bushelof soybean (60 pounds) is comprised of 48 pounds of protein meal and 11pounds of oil. While protein meal is the major component in soybean,oil, lecithin, tocopherols, isoflavones, etc. are all co-products andadd value to the bean. Soybean oil is the major edible oil used in theworld (40% of the 59.4 million metric tons in 1993). It also accountsfor 70% of the 14 billion pounds of edible vegetable oil in the UnitedStates. The primary food applications where the oil is used extensivelyare for baking and frying (40-45%), salad and cooking oil (40-45%),margarine and shortening (15-20%) and a wide spectrum of processedfoods. Development of other vegetable oils for specialty uses hasrecently affected the acreage and production of soybean. The low costand ready availability of soybean oil provide an excellent opportunityto upgrade this commodity item for specialty uses.

Food fats and oils are chemically composed of triesters of glycerolcontaining straight chain, normal aliphatic fatty acids, also referredto herein as triacylglycerols or triglycerides (TAG). The properties offood fats and oils are a reflection of the fatty acids contained in theTAG and their distribution on the glycerol backbone. When the meltingpoint of the TAG is below room temperature, the TAG is referred to as an“oil”. Triglycerides that melt above room temperature are referred to as“fat”. Gradients between fluidity and solidity exist. Partiallysolidified, non-pourable triglycerides are often referred to as “plasticfats”.

Fatty acids are organic acids having a hydrocarbon chain ranging inlength from about 4 to 24 carbons. Fatty acids differ from each other inchain length, and in the presence, number and position of double bonds.In cells, fatty acids typically exist in covalently bound forms, thecarboxyl portion being referred to as a fatty acyl group. The chainlength and degree of saturation of these molecules is often depicted bythe formula CX:Y, where “X” indicates number of carbons and “Y”indicates number of double bonds.

Typically, oil derived from commercial soybean varieties is composed ofapproximately 11% palmitic (C16:0), 4% stearic acid (C18:0), 21% oleicacid (C18:1), 56% linoleic acid (C18:2), and 10% linolenic acid (C18:3).The fatty acid composition of soybean oil, as well as all oils, largelydetermines its physical and chemical properties, and thus its uses.

Fatty acid biosynthesis has been the subject of research efforts in anumber of organisms. For reviews of fatty acid biosynthesis in plants,see Ohlrogge et al., (1995) Plant Cell, 7:957-970, Ohlrogge et al.,(1997) Annu Rev Plant Physiol Plant Mol Biol, 48:109-136 and Sommervilleet al. (1991) Science, 252:80-87.

As mentioned previously, the fatty acid composition of an oil determinesits physical and chemical properties, and thus its uses. Plants,especially plant species which synthesize large amounts of oils in plantseeds, for example soybean, are an important source of oils both foredible and industrial uses. Various combinations of fatty acids in thedifferent positions in the triglyceride will alter the properties of thetriglyceride. For example, if the fatty acyl groups are mostly saturatedfatty acids, then the triglyceride will be solid at room temperature. Ingeneral, however, vegetable oils tend to be mixtures of differenttriglycerides. The triglyceride oil properties are therefore a result ofthe combination of triglycerides which make up the oil, which are inturn influenced by their respective fatty acid compositions.

Plant breeders have successfully modified the yield and fatty acidcomposition of various plant seed oils by introducing desired traitsthrough plant crosses and selection of progeny carrying the desiredtrait forward. Application of this technique thus is limited to traitswhich are found within the same plant species. Alternatively, exposureto mutagenic agents can also introduce traits which may produce changesin the composition of a plant seed oil. However, it is important to notethat Fatty Acid Synthesis (FAS) occurs in most tissues of the plantincluding leaf (chloroplasts) and seed tissue (proplastids). Thus,although a mutagenesis approach can sometimes result in a desiredmodification of the composition of a plant seed oil, it is difficult toeffect a change which will not alter FAS in other tissues of the plant.

A wide range of novel vegetable oils compositions and/or improved meansto obtain or manipulate fatty acid compositions, from biosynthetic ornatural plant sources, are needed. Plant breeding, even withmutagenesis, cannot sufficiently meet this need and provide for theintroduction of novel oil.

For example, cocoa-butter has certain desirable qualities (mouthfeel,sharp melting point, etc.) which are a function of its triglyceridecomposition. Cocoa-butter contains approximately 24.4% palmitate (16:0),34.5% stearate (18:0), 39.1% oleate (18:1) and 2% linoleate (18:2).Thus, in cocoa butter, palmitate-oleate-stearate (POS) comprises almost46% of triglyceride composition, with stearate-oleate-stearate (SOS) andpalmitate-oleate-palmitate (POP) comprising the major portion of thebalance at 33% and 16%, respectively, of the triglyceride composition.Other novel oils compositions of interest might include trierucin (threeerucic) or a triglyceride with medium chain fatty acids in each positionof the triglyceride molecule.

Plant seed oils contain fatty acids acylated at the sn-1, sn-2, and sn-3positions of a glycerol backbone, referred to as a triacylglycerol(TAG). The structure of the TAG, as far as positional specificity offatty acids, is determined by the specificity of enzymes involved inacylating the fatty acyl CoA substrates to the glycerol backbone. Forexample, there is a tendency for such enzymes from many temperate andtropical crop species to allow either a saturated or an unsaturatedfatty acid at the sn-1 or the sn-3 position, but only an unsaturatedfatty acid at the sn-2 in the seed TAGs. In some species such as cocoa,TAG compositions suggest that this tendency is carried further in thatthere is an apparent preference for acylation of the sn-3 position witha saturated fatty acid, if the sn-1 position is esterified to asaturated fatty acid. Thus, there is a higher percentage of structuredTAG of the form Sat-Un-Sat (where Sat=saturated fatty acid andUn=unsaturated fatty acid).

Of particular interest are triglyceride molecules in which stearate isesterified at the sn-1 and sn-3 positions of a triglyceride moleculewith unsaturates in the sn-2 position particularly oleate. Vegetableoils rich in such SOS (Stearate-Oleate-Stearate) molecules share certaindesirable qualities with cocoa butter yet have a degree of additionalhardness when blended with other structured lipids. SOS-containingvegetable oils are currently extracted from relatively expensiveoilseeds from certain trees grown in tropical areas such as Sal, Shea,and Illipe trees from India, Africa, and Indonesia respectively. Cheaperand more conveniently grown sources for SOS-type vegetable oils aredesirable.

In addition, vegetable oils rich in stearate fatty acid content tend tobe solid at room temperature. Such vegetable fats can be used directlyin shortenings, margarine and other food “spread” products, obviatingthe need for chemical hydrogenation. Hydrogenation is a process wherebymolecular hydrogen is reacted with the unsaturated fatty acidtriglyceride until the desired degree of solidity is obtained. Thesolidity is commonly determined by the solid fat index (SFI, Officialand Tentative Methods, American Oil Chemists' Society, Cd 10-57(93),Champaign, Ill.). Values are determined by dilatometry (expansion involume) over a defined temperature range of 50°, 70°, 80°, 92° and 100°or 104° F. The hydrogenation process converts unsaturated fatty acids topartially or fully saturated fatty acids, and increases the heat andoxidative stability of the product. The iodine value (IV) measures thedegree of unsaturation of a fat. Lower values indicate greatersaturation. The oxidative stability may be measured by an oil stabilityindex (Official and Tentative Methods, American Oil Chemists' Society,Cd 1b-87, Champaign, Ill.) and active oxygen method (AOM, Official andTentative Methods, American Oil Chemists' Society, Cd 12h-92, Champaign,Ill.). The cost and any other factors associated with chemicalhydrogenation, such as the production of trans fatty acids, can beavoided if the vegetable oil is engineered to be stearate rich in theplant seed.

Moreover, some plant tissues use 18 carbon fatty acids as precursors tomake other compounds. These include saturated long chain fatty acidslonger than 18 carbons in length. Since very little stearate typicallyaccumulates in soybean plants, it may be necessary to increase stearateaccumulation if one wants to increase production of compounds whichdepend upon supply of stearate fatty acids for synthesis.

The fatty acid composition of soybean oil described above is oftenconsidered less than optimal in terms of oil functionality. While thelimitations of the fatty acid composition may be partly overcome bychemical hydrogenation, the trans fatty acids produced as a result ofthe hydrogenation process are Sat-Un-Satpected of having unfavorablehealth effects (Mensink, et al. (1990) N. Eng. J. Med. 323:439-445).

Through the efforts of traditional plant breeding techniques, the fattyacid composition of soybeans has been improved. For example, usingmutagenesis, plant breeders have been able to increase the amount ofstearate (C18:0) produced in the soybean oil. In such high stearatelines, designated as A6 (ATCC Accession No. 97392, Hammond and Fehr,(1983) Crop Science 23:192), stearate levels of up to about 25 weightpercent of the total fatty acid composition have been achieved. Suchhigh stearate containing lines have been further bred with mutantsoybean lines containing elevated levels of palmitate (16:0). Soybeanlines containing the elevated stearate levels produced by mutagenesisdemonstrate a negative correlation of increased stearate content andseed yield (Hartmann, et al. (1997) Crop Science 37:124-127). Attemptsto further increase the stearate content and/or improve the seed yieldof such increased stearate lines by breeding have thusfar provenunsuccessful.

List, et al. ((1996) J. Am. Oil. Chem. Soc. 73:729-732) describes theuse of genetically modified soybean oils in margarine formulations. Highstearate oil from soybean variety A6 was found to have an insufficientsolid fat index at 24.7° C. and higher temperatures to make margarine.The soybean oil was blended with cottonseed or soybean hardstocks toafford mixtures with sufficient solids content for formulation intomargarine.

While soybean based products are a major food source, improvements tothe nutritional and commercial quality of this product could add furthervalue to soybean based products. Alteration of the soybean oil contentand composition could result in products of higher nutritional contentand greater stability. The need for industrial hydrogenation ofpolyunsaturated oil for food applications could be reduced by thepreparation of soybean oil with increased concentrations of stearate.

SUMMARY OF THE INVENTION

The present invention is directed to methods for producing soybean oilhaving high levels of stearate (C18:0). The method of producing asoybean oil containing increased levels of stearate comprises expressionof an acyl-ACP thioesterase capable of producing C18:0 in the seedtissue of the soybean. In particular, the acyl-ACP thioesterase hassubstantial activity toward 18:0 acyl-ACP substrates, and preferably haslittle or no activity towards 16:0 acyl-ACP substrates.

The method generally comprises growing a soybean plant containing aconstruct comprising as operably linked components in the 5′ to 3′direction of transcription, a transcription initiation region functionalin a seed tissue and a DNA encoding an acyl-ACP thioesterase withsubstantial activity towards 18:0 acyl-ACP substrates and atranscription termination sequence.

The stearate content of the soybean oil preferably comprises greaterthan about 20%, more preferably greater than about 33% of the fatty acidmoieties in the oil. The oil of the present invention may be used as ablending source to make a blended oil product, or it may also be used inthe preparation of food.

In another embodiment of the present invention, a soybean oil having anincreased saturated fatty acid composition is provided. Soybean oilswith saturated fatty acid compositions of greater than 50 weight percentare exemplified herein.

In yet another embodiment of the instant invention, the novel soybeanoil, comprising the increased total saturated fatty acid compositions,provides a novel source of structured TAG of the Sat-Un-Sat(saturated-unsaturated-saturated) form.

The present invention further provides food products and methods fortheir preparation from a novel soybean [Glycine max] seed with increasedlevels of stearic and oleic acids, and decreased levels of linoleic andlinolenic acids under normal growing conditions. The novel soybean seedis produced by a soybean plant obtained from cross pollination of a highstearate plant with a low linolenate plant. There are multipleadvantages of a soybean seed with modified fatty acid content. Thesoybean oil has increased levels of stearic acid, normal levels ofpalmitic and oleic acids, and decreased levels of linoleic and linolenicacids relative to common soybean oil. Preferably, the soybean oil has astearic acid composition of above about 15%, a linoleic acid compositionbelow about 45%, and a linolenic acid composition below about 6%. Oilextracted from the soybean seeds possess increased stability andsuperior cooking characteristics than does oil extracted from standardsoybean seeds. The oil has higher levels of solids than does commonsoybean oil, making it a more preferred material for the preparation offood products such as margarine, tofu, soy flour, soymilk, andshortening. Interesterification of the oil can further enhance theamount of solids present, and the oil's utility in the preparation offood products. Food products prepared from modified soybeans displaycreamier textures than do food products prepared from common soybeans.While common and high stearate soybean oils require the addition ofhardstocks for the formation of margarines and other soy based products,the present oil may be used without the addition of adjuvants.

The novel soybean oil as well as the soybean seed containing the noveloil finds use in many applications.

DESCRIPTION OF THE FIGURES

FIG. 1. Nucleic acid and translated amino acid sequence of a mangosteenFatA-type acyl-ACP thioesterase clone (Garm FatA1) is provided. GarmFatA1 demonstrates primary thioesterase activity on 18:1 acyl-ACPsubstrate, but also demonstrates substantial activity on 18:0 substrate(approximately 10-20% of 18:1 activity), as well as little or noactivity on 16:0 substrates.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the subject invention, constructs and methods areprovided for the production of soybean plants with an increased level ofstearate (C18:0), as a percentage of the total fatty acids, in the seedoil. The methods for producing such soybean plants comprise transforminga soybean plant with expression constructs comprising a promotersequence functional in a plant seed operably linked to a DNA sequenceencoding a plant acyl-ACP thioesterase having substantial activitytoward 18:0-ACP substrates, preferably those with little or no activitytoward 16:0-ACP substrates (hereinafter referred to as stearoyl-ACPthioesterase), and a transcription termination sequence. The expressionconstructs provide an increase in the levels of stearate fatty acids inthe seed oil of the transformed soybean plants.

As described in more detail in the examples that follow, an acyl-ACPthioesterase coding sequence from mangosteen (Garcinia mangostana), GarmFatA1 (Hawkins and Kridl (1998) Plant Journal 13(6):743-752; and PCTPatent Application WO 96/36719, the entireties of which are incorporatedherein by reference) is used in expression constructs to generatetransgenic soybean plants with increased production of the stearoyl-ACPthioesterase in host cells. In particular the constructs are used todirect the expression of the Garm FatA1 thioesterase in plant seed cellsfor modification of triacylglycerol (TAG) fatty acid composition toprovide increased levels of C18:0 fatty acyl groups. Furthermore, theconstructs of the present invention may find use in plant geneticengineering applications in conjunction with plants containing elevatedlevels of C18:0 (stearate) fatty acids. Such plants may be obtained byantisense gene regulation of stearoyl-ACP desaturase using methodsdescribed by Knutzon et al (Proc. Nat. Acad. Sci. (1992) 89:2624-2628),and may also be obtained by co-suppression using sense expressionconstructs of the stearoyl-ACP desaturase gene, or by conventionalmutation and plant breeding programs. In addition, the constructs andmethods for increasing stearate in soybean seed may also find use inplant genetic engineering applications in conjunction with plantscontaining elevated levels of oleate (C 18:1) and/or decreased levels oflinoleate (C18:2) fatty acids and/or linolenate (18:3). Such plants withelevated levels of oleate and/or with decreased levels of linoleateand/or linolenate may be obtained through genetic engineering, or byconventional mutation and plant breeding programs.

A plant acyl-ACP thioesterase DNA sequence useful for the preparation ofexpression constructs for the alteration of stearate levels as describedherein encodes for amino acids, in the form of a protein, polypeptide orpeptide fragment, which amino acids demonstrate substantial activity on18:0 acyl-ACP substrates and little or no activity on 16:0-ACP to form18:0 free fatty acid (i.e., stearate) under plant enzyme reactiveconditions. By “enzyme reactive conditions” is meant that any necessaryconditions are available in an environment (i.e., such factors astemperature, pH, lack of inhibiting substances) which will permit theenzyme to function.

DNA sequences encoding for acyl-ACP thioesterase enzymes withsubstantial activity on 18:0 acyl-ACP substrates and little or noactivity on 16:0-ACP to form 18:0 free fatty acid (i.e., stearate) areknown in the art and are described in Hawkins and Kridl (1998) supra,and PCT Patent Application WO 96/36719. The Garm FatA1 DNA sequencedescribed therein and used herein demonstrates preferential activity onC18:1 acyl-ACP substrate, and also demonstrates substantial activity(approximately 25% of the 18:1 activity) on C18:0 acyl-ACP substrates.Only a small increase in C16:0 activity over activity in control cellsis observed, and the 16:0 activity represents only approximately 3% ofthe 18:1 activity.

In preparing the expression constructs, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate in the proper reading frame. Towardsthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resection, ligation, or the like may beemployed, where insertions, deletions or substitutions, e.g. transitionsand transversions, may be involved.

For the most part, the constructs will involve regulatory regionsfunctional in plants which provide for modified production of plantstearoyl-ACP thioesterase, and modification of the fatty acidcomposition. The open reading frame, coding for the plant stearoyl-ACPthioesterase or functional fragment thereof will be joined at its 5′ endto a transcription initiation regulatory region such as the wild-typesequence naturally found 5′ upstream to the thioesterase structuralgene, or to a heterologous regulatory region from a gene naturallyexpressed in plant tissues. Examples of useful plant regulatory generegions include those from T-DNA genes, such as nopaline or octopinesynthase, plant virus genes, such as CaMV 35S, or from native plantgenes.

For such applications when 5′ upstream non-coding regions are obtainedfrom other genes regulated during seed maturation, those preferentiallyexpressed in plant embryo tissue, such as ACP, napin and β-conglycinin7S subunit transcription initiation control regions, as well as theLesquerella hydroxylase promoter (described in Broun, et al. (1998)Plant Journal 13(2):201-210 and in U.S. patent application Ser. No.08/898,038) and the stearoyl-ACP desaturase promoter (Slocombe, et al.(1994) Plant Physiol. 104:1167-1176), are desired. Such “seed-specificpromoters” may be obtained and used in accordance with the teachings ofU.S. Pat. No. 5,420,034 having a title “Seed-Specific TranscriptionalRegulation” and in Chen et al., (1986), Proc. Natl. Acad. Sci.,83:8560-8564. Transcription initiation regions which are preferentiallyexpressed in seed tissue, i.e., which are undetectable in other plantparts, are considered desirable for fatty acid modifications in order tominimize any disruptive or adverse effects of the gene product.

Regulatory transcript termination regions may be provided in DNAconstructs of this invention as well. Transcript termination regions maybe provided by the DNA sequence encoding the plant stearoyl-ACPthioesterase or a convenient transcription termination region derivedfrom a different gene source, for example, the transcript terminationregion which is naturally associated with the transcript initiationregion. The skilled artisan will recognize that any convenienttranscript termination region which is capable of terminatingtranscription in a plant cell may be employed in the constructs of thepresent invention. As described herein, transcription terminationsequences derived from DNA sequences preferentially expressed in plantseed cells are employed in the expression constructs of the presentinvention.

The method of transformation is not critical to the instant invention;various methods of plant transformation are currently available. Asnewer methods are available to transform crops, they may be directlyapplied hereunder. For example, many plant species naturally susceptibleto Agrobacterium infection may be successfully transformed viatripartite or binary vector methods of Agrobacterium-mediatedtransformation. In addition, techniques of microinjection, DNA particlebombardment, and electroporation have been developed which allow for thetransformation of various monocot and dicot plant species.

In developing the DNA construct, the various components of the constructor fragments thereof will normally be inserted into a convenient cloningvector which is capable of replication in a bacterial host, e.g., E.coli. Numerous vectors exist that have been described in the literature.After each cloning, the plasmid may be isolated and subjected to furthermanipulation, such as restriction, insertion of new fragments, ligation,deletion, insertion, resection, etc., so as to tailor the components ofthe desired sequence. Once the construct has been completed, it may thenbe transferred to an appropriate vector for further manipulation inaccordance with the manner of transformation of the host cell.

Normally, included with the DNA construct will be a structural genehaving the necessary regulatory regions for expression in a host andproviding for selection of transformant cells. The gene may provide forresistance to a cytotoxic agent, e.g. antibiotic, heavy metal, toxin,etc., complementation providing prototrophy to an auxotrophic host,viral immunity or the like. Depending upon the number of different hostspecies in which the expression construct or components thereof areintroduced, one or more markers may be employed, where differentconditions for selection are used for the different hosts. A number ofmarkers have been developed for use for selection of transformed plantcells, such as those which provide resistance to various antibiotics,herbicides, or the like. The particular marker employed is not essentialto this invention, one or another marker being preferred depending onthe particular host and the manner of construction.

As mentioned above, the manner in which the DNA construct is introducedinto the plant host is not critical to this invention. Any method whichprovides for efficient transformation may be employed. Various methodsfor plant cell transformation include the use of Ti- or Ri-plasmids,microinjection, electroporation, DNA particle bombardment, liposomefusion, or the like. In many instances, it will be desirable to have theconstruct bordered on one or both sides by T-DNA, particularly havingthe left and right borders, more particularly the right border. This isparticularly useful when the construct uses A. tumefaciens or A.rhizogenes as a mode for transformation, although the T-DNA borders mayfind use with other modes of transformation.

Various methods of transforming cells of soybean have been previouslydescribed. Examples of soybean transformation methods have beendescribed, for example, by Christou et al. U.S. Pat. No. 5,015,580 andby Hinchee et al. U.S. Pat. No. 5,416,011, the entireties of which areincorporated herein by reference.

Once a transgenic plant is obtained which is capable of producing seedhaving a modified fatty acid composition, traditional plant breedingtechniques, including methods of mutagenesis, may be employed to furthermanipulate the fatty acid composition. Alternatively, additional foreignfatty acid modifying DNA sequence may be introduced via geneticengineering to further manipulate the fatty acid composition.

One may choose to provide for the transcription or transcription andtranslation of one or more other sequences of interest in concert withthe expression of a plant stearoyl-ACP thioesterase in a plant hostcell. In particular, the reduced expression of stearoyl-ACP desaturasein combination with expression of a plant stearoyl-ACP thioesterase maybe preferred in some applications.

When one wishes to provide a plant transformed for the combined effectof more than one nucleic acid sequence of interest, typically a separatenucleic acid construct will be provided for each. The constructs, asdescribed above contain transcriptional or transcriptional andtranslational regulatory control regions. The constructs may beintroduced into the host cells by the same or different methods,including the introduction of such a trait by the inclusion of twotranscription cassettes in a single transformation vector, thesimultaneous transformation of two expression constructs,retransformation using plant tissue expressing one construct with anexpression construct for the second gene, or by crossing transgenicplants via traditional plant breeding methods, so long as the resultingproduct is a plant having both characteristics integrated into itsgenome.

By decreasing the amount of stearoyl-ACP desaturase, an increasedpercentage of saturated fatty acids is provided. Using anti-sense,transwitch, ribozyme or some other stearoyl-ACP desaturase reducingtechnology, a decrease in the amount of stearoyl-ACP desaturaseavailable to the plant cell is produced, resulting in a higherpercentage of saturates such as one or more of stearate (C18:0),arachidate (C20:0), behenate (C22:0) and lignocerate (C24:0). Inrapeseed reduced stearoyl-ACP desaturase results in increased stearatelevels and total saturates (Knutzon et al. (1992) Proc. Nat. Acad. Sci.89:2264-2628).

Of special interest is the production of triglycerides having increasedlevels of stearate. In addition, the production of a variety of rangesof stearate is desired. Thus, plant cells having lower and higher levelsof stearate fatty acids are contemplated. For example, fatty acidcompositions, including oils, having a 10% level of stearate as well ascompositions designed to have up to an approximate 60% level of stearateor other such modified fatty acid(s) composition are contemplated.

As described in more detail in the examples that follow, constructs areprepared to direct the expression of a stearoyl-ACP thioesterase inplant seed tissue. Such expression constructs allow for the increase in18:0 levels in oils obtained from the seeds of transformed soybeanplants.

Increases in the levels of stearate in soybeans transformed to expressGarm FatA1 range from 4 fold to approximately 13 fold over the levelsobtained in seeds from nontransformed control plants. Additionally, bydecreasing the amount of stearoyl-ACP desaturase available to the plantFAS complex in conjunction with an increase of the amount ofstearoyl-ACP thioesterase available, a more marked increased percentageof stearate may be obtained. By manipulation of various aspects of theDNA constructs (e.g., choice of promoters, number of copies, etc.) andtraditional breeding methods, one skilled in the art may achieve evengreater levels of stearate. By expression of a plant stearoyl-ACPthioesterase in seed tissue or a decrease in the expression ofstearoyl-ACP desaturase or a combination of both, an increasedpercentage of stearate can be achieved in soybean. In addition, modifiedthioesterase encoding DNA sequences may find use for increasing stearatelevels in seed tissue. Such modified thioesterase sequences may beobtained as described in PCT Patent Application WO 96/36719.

Surprisingly, in the oil of seeds from T2 soybean lines transformed toexpress the Garm FatA1 DNA sequence from the β-conglycinin 7S subunitpromoter, stearate levels of up to 53 percent as a percentage of thetotal fatty acid composition are obtained. In addition, in the oil ofthe initial transformed lines expressing Garm FatA1 DNA sequence fromthe β-conglycinin 7S subunit promoter increases in the levels ofstearate obtained from individual seeds range from about 14 weightpercent up to about 53 weight percent. Furthermore, transgenic soybeanplants expressing the Garm FatA1 DNA sequence from the napin derivedpromoter accumulate increased levels of stearate ranging fromapproximately 20 weight percent up to approximately 45 weight percent inindividual seeds of T2 soybean lines. Stearate levels obtained from theoil of individual seeds of nontransformed control soybeans range fromapproximately 4 weight percent to approximately 6 weight percent.Preferred oil compositions for many applications include 33 weightpercent or greater stearate fatty acids as a component of the soybeanoil.

In addition, transformed soybean lines containing increased stearatelevels of the present invention also demonstrate an increase in thetotal levels of saturated fatty acids. Transformed soybean linescontaining elevated stearate levels also contain increased levels ofArachidic acid (20:0) and Behenic acid (22:0). Increases in 20:0 rangefrom about 3 fold to about 11 fold over the levels of 20:0 obtained fromthe seed oil of nontransgenic control soybean lines. Increases in 22:0range from about 2 fold to about 5 fold over the levels of 22:0 obtainedfrom the seed oil of nontransgenic control soybean lines.

Thus, in soybean lines transformed to express a stearoyl-ACPthioesterase in the seed tissue, total saturated fatty acids (16:0,18:0, 20:0 and 22:0) comprise at least 30 percent of the total fattyacids as a percentage of weight, preferably above 50 weight percent. Insome cases, total saturated fatty acid levels of above about 65 weightpercent may be obtained.

The novel soybean oil compositions of the present invention compriseincreased total saturated fatty acids and provide a novel source ofstructured TAG of the Sat-Un-Sat form. For oil compositions havinggreater than about 33 weight percent stearate the Sat-Un-Sat form of TAGmay comprise 25 percent or greater of the total TAG composition as astearate-unsaturated-stearate form of TAG. It is apparent that byutilizing a high oleic acid soybean line that one may produce a soybeanoil with a high proportion of a stearate-oleate-stearate form of TAG. Anexample of such high oleic acid soybean oil is described in PCTApplication WO 97/40698.

The present invention further provides food products and methods fortheir preparation from a novel soybean [Glycine max] seed with increasedlevels of stearic and oleic acids, and decreased levels of linoleic andlinolenic acids under normal growing conditions. The novel soybean seedis produced by a soybean plant obtained from cross pollination of a highstearate plant with a low linolenate plant. There are multipleadvantages of a soybean seed with modified fatty acid content. Thesoybean oil has increased levels of stearic acid, normal levels ofpalmitic and oleic acids, and decreased levels of linoleic and linolenicacids relative to common soybean oil. Preferably, the soybean oil has astearic acid composition of above about 15%, a linoleic acid compositionbelow about 45%, and a linolenic acid composition below about 6%. Oilextracted from the soybean seeds possess increased stability andsuperior cooking characteristics than does oil extracted from standardsoybean seeds. The oil has higher levels of solids than does commonsoybean oil, making it a more preferred material for the preparation offood products such as margarine, tofu, soy flour, soymilk, andshortening. Interesterification of the oil can further enhance theamount of solids present, and the oil's utility in the preparation offood products. Food products prepared from modified soybeans displaycreamier textures than do food products prepared from common soybeans.While common and high stearate soybean oils require the addition ofhardstocks for the formation of margarines and other soy based products,the present oil may be used without the addition of adjuvants.

The soybean oil compositions of the present invention containing novelfatty acid compositions may find use in a number of applications,without the need for chemical modifications prior to use as describedherein, or as described in PCT application titled “Food ProductsContaining Structured Triglycerides”, PCT/US97/06037, the entirety ofwhich is incorporated herein by reference. The soybean oil of thepresent invention may find use in the preparation of foods to facilitatecooking or heating applications.

The soybean oils produced by the methods of the present invention may beused in the formation of emulsions comprising water and soybean oil. Thesoybean oil may be treated by interesterification prior to the formationof an emulsion. As used herein, interesterification refers to theprocess of rearranging the glyceride structure of fats.Interesterification is accomplished by a chemical reaction in whichfatty acids are rearranged on the glycerol molecule without modificationof the fatty acids themselves. An emulsion may preferably comprisebetween about 70% and about 90% by volume soybean oil, and between 10%and about 30% by volume water. The aqueous emulsion may further bedefined as margarine. As used herein, the term “margarine” refers to anedible emulsion comprising oil and water that is both solid andspreadable at 25° C.

Alternatively, the soybean seeds containing the modified fatty acidcompositions of the present invention, may be used to prepare soymilk.Soymilk may be prepared by the steps of selecting soybean seeds,contacting the seeds with water to form a mixture, heating the mixture,grinding the mixture, and removing the solids to form soymilk. Removalof solids may be accomplished by methods including, but not limited to,filtration, sedimentation and centrifugation. The heating step maycomprise heating the mixture to any temperature suitable for theformation of tofu, preferably to a temperature sufficient to inactivatethe trypsin inhibitor in the liquid at least about 80% as compared tothe trypsin inhibitor activity prior to heating, and most preferably toa temperature between about 90° and about 100° C. Trypsin inhibitoractivity may be conveniently assayed using colorimetric method describedin Liu and Markakis ((1989), Cereal Chem 66(5):415-422).

Soybean seeds containing the novel soybean oil compositions of thepresent invention may find use in the preparation of tofu. Tofu may beprepared by the steps of selecting soybean seeds containing soybean oilhaving oil compositions of the present invention, contacting the seedswith water to form a mixture, heating the mixture, grinding the mixture,removing the solids to form a filtrate, adding a coagulant, and coolingthe filtrate to form tofu. The coagulant used may be, but is notlimited, to glucono-δ-lactone, lemon juice, sea salt, calcium sulfate ormagnesium chloride. Removal of solids may be accomplished by methodsincluding, but not limited to filtration, sedimentation, andcentrifugation. The heating step may comprise heating the mixture to anytemperature suitable for the formation of tofu, preferably to atemperature sufficient to inactivate the trypsin inhibitor in the liquidat least about 80% as compared to the trypsin inhibitor activity priorto heating, and most preferably to a temperature between about 90° andabout 100° C. Trypsin inhibitor activity may be conveniently assayedusing colorimetric method described in Liu and Markakis ((1989), CerealChem 66(5):415-422). The cooling step may comprise cooling the filtrateto any temperature suitable for the formation of tofu, and morepreferably to between about 0° C. and about 25° C.

Soy flour may also be prepared from soybean seeds containing the oilcompositions of the present invention. A method for preparation of soyflour comprises the steps of selecting soybean seeds and grinding theseeds to produce soy flour. Preferably, the soybean seeds containsoybean oil having increased levels of stearate. The grinding step maybe performed by any means suitable for the production of soy flour,including, but not limited to, grinding with wheels, mortar and pestle,plates and blades.

The soybean oil of the present invention may be further used to prepareshortenings. As used herein, the term “shortening” refers to fats,usually plastic in nature, that provide functional effects related tostructure, texture and the eating qualities of a variety of foodproducts. The shortening prepared based on the soybean oil of thepresent invention may also include the incorporation of emulsifiers orsurfactants (selected from 21 C.F.R. 172), such emulsifiers used toprovide additional functional effects in the final food product. Theshortening based on the soybean oil of the present invention may alsoinclude a variety of hardstocks (fully hydrogenated triglyceridessourced from a variety of common food oils) that could be used to modifyor augment the SFC of the final product and, hence, the physicalproperties and functionality of the final blend. The plasticity andcrystal structure of the final shortening composition based on thesoybean oil of the present invention, whether emulsified or not, may befurther modified through the process of controlled crystallization andspecific gravity reduction known as votation. In this process, themolten shortening described variously above is fed through ascraped-surface heat exchanger, and crystallized in a directedmanner-usually in the most functional crystalline form. During thisprocess, a gas is usually whipped into the solidifying product to adjustthe specific gravity of the shortening product, and hence itsplasticity. This is done, primarily, to enhance the handlingcharacteristics of the final shortening and to allow it to be betterincorporated into a variety of food product systems. The emulsifier maygenerally be any material suitable for the preparation of shortening,preferably an emulsifier approved as a food additive per 21 C.F.R. §172, and more preferably is a monoglyceride. The use of the novelsoybean oil of the present invention may allow for a reduced level ofemulsifier addition to achieve the same functional effects as would berequired in a standard soybean oil based shortening arrived at throughhydrogenation to attain equal solids.

The soybean seeds, oil, and products therefrom may also find use in anumber of additional applications known to the art, including the use invarious animal feed applications.

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are included forpurposes of illustration only and are not intended to limit the presentinvention.

EXAMPLES Example 1 Plant Expression Vector Construction

Plant vectors are constructed to control the expression of a member ofthe FatA class of acyl-ACP thioesterases from Garcinia mangostana (GarmFatA1, Hawkins and Kridl (1998) supra, and PCT Patent Application WO96/36719) in seeds of soybean utilizing different seed enhancedpromoters.

A plant transformation construct, pWRG5374, is prepared to express GarmFatA1 in the embryo tissue of the soybean seed utilizing the napinpromoter. A DNA fragment containing the napin 5′/Garm FatA1/napin 3′,described in Hawkins and Kridl (1998) supra, is cloned into a vectorcontaining the selectable marker β-glucuronidase (GUS, Jefferson et al.,Proc. Natl. Acad. Sci. (1986) 83:8447-8451) driven by the CAMV 35S(Gardner, et al. (1981) Nucleic Acids Res. 9:2871-2888) promoter. TheGUS gene contains an untranslated leader sequence derived from a soybeanribulose-bis-phosphate carboxylase (RuBisCo) small subunit,(Grandbastien, et al. (1986) Plant Mol. Biol. 7:451-466), ssuL and atranslational termination sequence derived from the soybean RuBisCo,(Berry-Lowe (1982) Jour. Mol. Appl. Genet. 1:483-498), SpA. Examples ofvectors utilizing a GUS selectable marker are described in EuropeanPatent 0 301 749 B1, the entirety of which is incorporated herein byreference. The resulting expression construct, pWRG5374, contains thenapin 5′/Garm FatA1/napin 3′ sequences as well as the 35S-ssuL/GUS/SpA3′ for transgenic selection by indigo blue staining.

The soybean transformation construct, pWRG5378, containing the GarmFatA1 coding sequence expressed from the β-conglycinin 7S subunitpromoter was prepared as follows. The Garm FatA1 coding sequence andnapin 3′ poly-A termination sequences were obtained from plasmidpCGN5253 (described in Hawkins and Kridl (1998) supra). A soybeanexpression plasmid pWRG5375 was constructed by insertion of the GarmFatA1 coding and napin 3′ sequences downstream of a heterologouspromoter from the soybean α′ subunit of β-conglycinin (soy 7s, (Chen etal., (1986), Proc. Natl. Acad. Sci., 83:8560-8564)). A 941 bp BamHI-XhoIfragment containing the soy 7s promoter was ligated with a 5186 bpfragment from plasmid pCGN5253 produced by partial digestion with KpnIand complete digestion with SalI. Additionally, an 8 bp BamHI-KpnIadapter having the DNA sequence 5′-GATCGTAC-3′ was used to fuse theBamHI site from the soy 7s promoter fragment with the KpnI site frompCGN5253. The resulting plasmid was named pWRG5375. A 3477 bp SacIfragment from plasmid pWRG5375 containing the soy 7s/Garm FatA1/napin 3′was ligated to a 6135 bp fragment containing the β-glucuronidase (GUS)marker cassette (described above) for selection of transgenic soybeanplants. The soy 7s/Garm FatA1/napin 3′ cassette was inserted such thatthe transcription of the GUS gene was in the same direction as that ofthe Garm FatA1 coding sequence. The resulting 8329 bp plasmid wasdesignated as pWRG5378.

Example 2 Soybean Transformation with Garm FatA1 Constructs

Plasmids pWRG5374 and pWRG5378 were digested with NotI and linearizedfragments containing both the chimeric Garm FatA1 coding sequence andGUS expression cassettes were purified by HPLC. The linear DNA fragmentswere stably introduced into soybean (Asgrow variety A5403) by the methodof McCabe, et. al. (1988) Bio/Technology 6:923-926.

Transformed soybean plants are identified by indigo blue staining ofseed tissue with 1 mM X-Gluc (Clontech), 0.1M NaPO₄ (pH 7.0), 0.5 mMpotassium ferrocyanide.

Example 3 Fatty Acid Compositional Analysis

Fatty acid compositions were analyzed from seed of soybean linestransformed with pWRG5374 or pWRG5378. One to five seeds of each of thetransgenic and control soybean lines were ground individually using atissue homogenizer (Pro Scientific) for oil extraction. Oil from groundsoybean seed was extracted overnight in 1.5 ml heptane containingtriheptadecanoin (0.50 mg/ml). Aliquots of 200 μl of the extracted oilwas derivatized to methyl esters with the addition of 500 μl sodiummethoxide in absolute methanol. The derivatization reaction was allowedto progress for 20 minutes at 50° C. The reaction was stopped by thesimultaneous addition of 500 μl 10% (w/v) sodium chloride and 400 μlheptane. The resulting fatty acid methyl esters extracted in hexane wereresolved by gas chromatography (GC) on a Hewlett Packard model 6890 GC.The GC was fitted with a Supelcowax 250 column (30 m, 0.25 mm id, 0.25micron film thickness) (Supelco, Bellefonte, Pa.). Column temperaturewas 175° C. at injection and the temperature programmed from 175° C. to245° C. to 175° C. at 40° C./min. Injector and detector temperatureswere 250° C. and 270° C., respectively.

The results of the fatty acid compositional analysis from seed oil ofthe initial transformed 5374 soybean lines is provided in Table 1.Averages are provided where oil compositional analysis was performed onmore than one seed from the initial transformant. In seed of transgenicsoybean plants expressing Garm FatA1 from the napin promoter, stearate(C18:0) levels were significantly increased over the levels obtainedfrom the seed oil of nontransformed control plants. The increase instearate is primarily at the expense of oleate, and to a lesser degreelinoleate and palmitic all of which were decreased in the transgeniclines. In addition, increases in all saturates examined greater thanC18:0 were observed.

TABLE 1 STRAIN ID GUS %16:0 %18:0 %18:1 %18:2 %18:3 %20:0 %22:05374-A5403-3 + 6.71 24.88 14.53 43.73 6.99 1.84 1.03 5374-A5403-3 + 6.6226.52 11.9 44.89 7.15 1.8 0.9 5374-A5403-3 + 7.59 22.99 13.17 45.32 8.191.63 0.84 5374-A5403-3 + 7.28 23.1 13 43.65 9.74 1.88 1.135374-A5403-3 + 7.39 26.54 8.4 41.95 12.38 1.9 1.07 AVERAGE 7.12 24.8112.2 43.91 8.89 1.81 0.99 5374-A5403-4 + 7.38 20.81 16.45 46.61 5.991.57 0.87 5374-A5403-4 + 7.96 18.28 14.69 47.93 8.29 1.54 0.965374-A5403-4 + 10.02 9.64 22.78 49.41 6.02 0.91 0.79 5374-A5403-4 + 9.2412.54 23.06 45.97 5.9 1.17 0.8 5374-A5403-4 + 7.41 20.07 13.42 45.9110.32 1.55 0.97 AVERAGE 8.40 16.27 18.08 47.17 7.29 1.35 0.885374-A5403-14 + 7.96 38.39 7.82 37.34 6.23 2.41 1.07 5374-A5403-35 9.2433.53 11.14 38.15 7.54 1.97 0.9 5374-A5403-36 + 8.18 19.37 13.92 47.058.47 1.6 1.06 5374-A5403-36 + 7.5 19.99 13.49 47.15 8.99 1.59 0.955374-A5403-36 + 7.05 23.44 10.54 46.19 9.64 1.82 0.99 5374-A5403-36 +7.83 20.06 13.53 47.27 8.45 1.57 0.94 5374-A5403-36 + 7.49 22.9 11.8146.14 8.54 1.72 1.03 AVERAGE 7.61 21.15 12.66 46.76 8.82 1.66 0.995374-A5403-172 + 7.92 15.53 29.9 38.2  5.8 1.36 0.89 5374-A5403-172 +7.37 22.45 16.63 44.3  6.58 1.56 0.77 5374-A5403-172 + 8.74 14.38 20.1747.33 6.94 1.17 0.84 AVERAGE 8.01 17.45 22.23 43.28 6.44 1.36 0.83Control A5403 − 11.62 4.3 24.26 49.84 7.47 0.48 0.57 A5403 − 12.32 4.2421.93 52.32 7.49 0.44 0.46 A5403 − 12.64 4.25 20.49 53.42 7.81 0.43 0.51A5403 − 12.17 4.22 21.56 .52.48  8.15 0.44 0.51 A5403 − 11.68 4.32 25.6849.67 7.06 0.48 0.54 AVERAGE 12.09 4.27 22.78 51.55 7.60 0.45 0.52

Selected T2 lines also show the trends of increased stearate, anddecreased palmitate, oleate and linoleate levels in the seed oil (Table2). Furthermore, in seed of T2 5374 soybean lines (T3 seed), stearatelevels as high as approximately 45% of the fatty acid methyl esters areobserved. These levels are increased from approximately 34% in the T1generation. While null progeny which do not contain the Garm FatA1transgene contain approximately 4.5% of the fatty acid methyl esters asstearate.

TABLE 2 STRAIN ID GUS 16:0 18:0 18:1 18:2 18:3 20:0 22:05374-A5403-3-417 + 6.57 37.18 7.39 35.38 9.36 2.68 1.25374-A5403-3-417 + 6.52 38.66 8.26 33.82 8.43 2.78 1.295374-A5403-3-417 + 6.23 39.26 7.26 35.11 7.84 2.8 1.265374-A5403-3-417 + 6.75 33.55 8.91 37.69 9.13 2.51 1.195374-A5403-3-417 + 6.18 42.21 5.88 33.36 7.99 2.94 1.23 average 6.4538.17 7.54 35.07 8.55 2.74 1.23 5374-A5403-35-483 + 5.78 45.64 6.3 31.866.15 2.95 1.16 5374-A5403-35-483 + 5.82 38.21 7.83 37.06 7.22 2.54 1.125374-A5403-35-483 + 5.84 38.37 7.44 37.91 6.43 2.59 1.225374-A5403-35-483 + 5.74 41.56 6.31 35.4 6.98 2.71 1.125374-A5403-35-483 + 5.58 40.35 7.06 36.91 6.11 2.63 1.16 average 5.7540.83 6.99 35.83 6.58 2.68 1.16 5374-A5403-172-401 + 6.73 23.12 15.0246.04 6.05 1.77 0.97 5374-A5403-172-401 + 6.92 21.96 14.85 46.47 6.71.78 1.02 5374-A5403-172-401 + 6.49 24.15 14.11 45.74 6.46 1.83 0.965374-A5403-172-401 + 6.83 23.09 13.64 46.56 6.85 1.79 0.965374-A5403-172-401 + 8.32 20.32 11.94 47.05 9.36 1.69 1.02 average 7.0622.53 13.91 46.37 7.08 1.77 0.99 5374-A5403-36-353 + 6.18 30.73 11.3641.3 7.3 1.92 0.89 5374-A5403-36-353 + 6.42 30.82 11.14 41.03 7.53 1.850.85 5374-A5403-36-353 + 6.43 30.03 11.61 40.84 8.12 1.84 0.845374-A5403-36-353 + 6.66 29.27 13.98 40.82 6.26 1.77 0.835374-A5403-36-353 + 6.15 30.32 13.67 40.76 5.95 1.92 0.89 average 6.3730.23 12.35 40.95 7.03 1.86 0.86 5374-A5403-36-489 + 6.57 34.87 10.5637.1 7.23 2.34 1.09 5374-A5403-36-489 + 6.25 37.1 8.43 37.65 7.05 2.33 15374-A5403-36-489 + 6.36 36.22 10.68 36.18 6.88 2.39 1.085374-A5403-36-489 + 6.29 36.28 8.69 38.06 7.08 2.33 1.045374-A5403-36-489 + 6.26 36.6 8.33 37.79 7.25 2.44 1.11 average 6.3536.21 9.34 37.36 7.10 2.37 1.06 Control 5374-A5403-36-341 − 10.7 6 24.4650.9 6.29 0.53 0.61 5374-A5403-36-341 − 11.2 4.92 20.68 54.35 7.37 0.470.57 5374-A5403-36-341 − 11.27 4.27 23.71 52.72 6.55 0.43 0.535374-A5403-36-341 − 11.33 4.78 20.4 54.38 7.58 0.46 0.555374-A5403-36-341 − 11.55 4.52 18.59 55.07 8.69 0.46 0.56 null segregantAve 11.21 4.90 21.57 53.48 7.30 0.47 0.56

The results of the fatty acid compositional analysis for transformed5378 soybean plants are shown in Table 3. Seeds of soybean plantstransformed to express Garm FatA1 from the 7S promoter producedincreased levels of stearate over those levels observed in seeds ofnontransformed control plants. In the seed oil of some T1 5378transgenic soybean, stearate levels of as high as approximately 53% ofthe fatty acids were obtained, while levels of approximately 4% wereobserved in nontransformed control plants.

TABLE 3 STRAIN ID GUS %16:0 %18:0 %18:1 %18:2 %18:3 %20:0 %22:05378-A5403-28 + 6.27 41.81 8.64 34.22 5.13 2.62 1 5378-A5403-28 + 6.342.63 10.15 32.76 4.16 2.55 0.87 5378-A5403-28 + 6.48 43.11 7.47 33.725.3 2.61 1.04 5378-A5403-28 + 6.48 43.12 9.32 32.97 4.14 2.64 1.02AVERAGE 6.38 42.67 8.90 33.42 4.68 2.61 0.98 5378-A5403-48 + 8.19 25.5112.37 44.21 6.85 1.62 0.88 5378-A5403-48 + 7.74 33.77 12.13 36.33 6.412.23 1.05 5378-A5403-48 + 7.42 40.06 9.82 30.97 7.26 2.84 1.235378-A5403-48 + 7.89 45.26 5.73 30.23 6.8 2.71 1.06 5378-A5403-48 + 7.0447.2 5.69 29.58 6.13 2.86 1.2 AVERAGE 7.66 38.36 9.15 34.26 6.69 2.451.08 5378-A5403-59 + 9.96 47.1 9.17 22.11 4.12 4.78 2.3 5378-A5403-59 +7.06 47.3 4.44. 29.02 7.17 3.31 1.44 5378-A5403-59 + 11.72 50.5 4.6920.86 5.35 4.34 1.93 5378-A5403-59 + 7.55 51.95 4.99 24.53 5.02 4 1.72AVERAGE 9.07 49.21 5.82 24.13 5.42 4.11 1.85 5378-A5403-60 + 7.7 35.26.09 37.85 8.77 2.74 1.34 5378-A5403-60 + 7.19 35.53 5.86 38.36 8.572.79 1.41 5378-A5403-60 + 7.27 36.4 5.51 37.78 8.61 2.78 1.355378-A5403-60 + 9.01 52.21 1.71 23.14 7.62 4.41 1.59 5378-A5403-60 +9.83 52.94 1.77 21.85 7.59 4.28. 1.42 AVERAGE 8.2 42.46 4.19 31.80 8.233.40 1.42 5378-A5403-69 + 11.67 4.67 18.46 55.85 7.93 0.43 0.475378-A5403-69 + 8.69 13.65 19.02 48.86 7.19 1.13 0.9 5378-A5403-69 +8.13 18.5 14.7 48.52 7.4 1.41 0.83 5378-A5403-69 + 7.1 21.26 12.86 48.937.1 1.43 0.89 5378-A5403-69 + 8.04 44.02 8.09 28.04 7.34 2.96 1.16AVERAGE 8.73 20.42 14.63 46.04 7.39 1.47 0.85 5378-A5403-103 + 9.3140.01 8.73 30.52 7.08 2.85 1.04 5378-A5403-113 + 7.41 49.06 4.91 28.835.45 2.91 1.06 5378-A5403-113 + 11.01 49.44 4.88 22.53 7.15 3.24 1.15378-A5403-113 + 7.03 49.79 4.09 29.08 5.9 2.79 1 5378-A5403-113 + 8.3251.04 4.29 27.06 5.1 2.91 0.92 5378-A5403-113 + 8.52 52.59 3.69 26.354.64 2.93 0.86 AVERAGE 8.46 50.38 4.37 26.77 5.65 2.96 0.99 ControlA5403 − 11..62 4.3 24.26 49.84 7.47 0.48 0.57 A5403 − 12.32 4.24 21.9352.32 7.49 0.44 0.46 A5403 − 12.64 4.25 20.49 53.42 7.81 0.43 0.51 A5403− 12.17 4.22 21.56 52.48 8.15 0.44 0.51 A5403 − 11.68 4.32 25.68 49.677.06 0.48 0.54 AVERAGE 12.09 4.27 22.78 51.55 7.60 0.45 0.52

In T3 seed of selected T2 soybean lines, increases in stearate of ashigh as approximately 53% of the total fatty acid composition wereobtained (Table 4), similar to those levels obtained from seed oil fromT1 5378 soybean lines. Furthermore, similar to the 5374 soybean plants,decreases in palmitate, oleate and linoleate were observed in both theT2 and T3 seed oil. In addition, increases in saturates greater thanC18:0 are also obtained in both the T2 and T3 generations.

TABLE 4 STRAIN ID GUS 16:0 18:0 18:1 18:2 18:3 20:0 22:05378-A5403-48-269 + 8.63 50.62 5.5 23.07 7.4 3.34 1.145378-A5403-48-269 + 8.27 53.31 4.54 23.14 6.2 3.26 1.095378-A5403-48-269 + 8.6 51.92 4.84 22.65 7.27 3.43 1.015378-A5403-48-269 + 8.62 51.62 4.39 23.2 7.43 3.46 1.065378-A5403-48-269 + 9.07 50.57 4.77 22.61 7.97 3.52 1.22 average 8.6451.61 4.81 22.93 7.25 3.40 1.10 5378-A5403-113-304 + 6.99 49.4 4.2329.37 6.12 2.73 0.98 5378-A5403-113-304 + 6.79 50.25 3.77 28.53 6.762.74 0.99 5378-A5403-113-304 + 6.79 50.19 3.73 28.8 6.61 2.75 0.985378-A5403-113-304 + 6.34 47.67 4.08 30.03 7.96 2.77 1.015378-A5403-113-304 + 6.81 49.89 3.93 29.16 6.43 2.63 0.92 average 6.7449.48 3.95 29.18 6.78 2.72 0.98 5374-A5403-36-341 − 10.7 6 24.46 50.96.29 0.53 0.61 5374-A5403-36-341 − 11.2 4.92 20.68 54.35 7.37 0.47 0.575374-A5403-36-341 − 11.27 4.27 23.71 52.72 6.55 0.43 0.535374-A5403-36-341 − 11.33 4.78 20.4 54.38 7.58 0.46 0.555374-A5403-36-341 − 11.55 4.52 18.59 55.07 8.69 0.46 0.56 null segregantAve 11.21 4.90 21.57 53.48 7.30 0.47 0.56

The above results demonstrate that by expression of an acyl-ACPthioesterase with substantial activity towards 18:0 acyl-ACP substrates,and capable of producing C18:0 in seed tissue of soybean plants, it isnow possible to increase the levels of stearate in the seed oil ofsoybean.

Example 4 Composition of High Stearate Soybean Oil

In this particular case, soybean variety Hartz H4152 (also known asHS-2) was developed with a unique fatty acid composition of about 24% orabove stearate and below about 3%, preferably about 2.5% linolenate.H4152 was derived from the cross between a soybean line with highstearate content (H90-127-113, also known as HS-1) and a line with lowlinolenic content (N85-2176). H90-127-113 is a Hartz variety derivedfrom a cross between Hartz variety H5668 and soybean line A6. A6 is asoybean mutant with high stearate seed content (28.1%) released in 1981by Iowa State University (Hammond, E. G. and W. R. Fehr. 1983.Registration of A6 germplasm line of soybean. Crop Sci. 23: 192-193).The high stearate content in line A6 has been determined to beconditioned by a single recessive gene, fas-a (Graef, G. L., W. R. Fehr,and E. G. Hammond. 1985. Inheritance of three stearic acid mutants ofsoybean. Crop Sci. 25: 1076-1079). N85-2176 is a release from NorthCarolina State University selected for its low linolenate seed content.

The F₁ seeds from the cross between H90-127-113 and N85-2176 were grownin the greenhouse in November and December. In February, small portionsof the F₂ seeds opposite the embryo were removed and analyzed in thelaboratories of Hartz Seed Co. for fatty acid composition using a gaschromatography. F₂ seed number 27 was selected for its high stearate andlow linolenate levels and was grown in the greenhouse. The F₃ seed fromplant #27 were planted in the field at Stuttgart, AR in the followingsummer. Ten agronomically desirable F₃ plants with desirable fatty acidcomposition were selected, and F₄ progeny rows from those plants wereplanted at Stuttgart in the summer. Twenty agronomically desirable,uniform single plants were selected from row number 4. The F₅ progeny ofthose 20 plants was planted in single rows in a winter nursery in SantaIsabel, Puerto Rico. Ten uniform, single rows were harvested and bulked.The resulting seed was grown in a 0.2 acre breeder increase atStuttgart, AR during the following summer, forming the foundation forHartz variety H4152.

TABLE 5 Fatty acid distributions of soybean oil Palmitic Stearic OleicLinoleic Linolenic Soybean oil C16:0 C18:0 C18:1 C18:2 C18:3 Common 11 423 53 8 High stearate 10 21 22 41 3 (H4152)

The composition of the high stearate soybean oil is unique in that ithas low linoleic and linolenic and high oleic and stearic fatty acids.This improves the stability of the oil to oxidative degradation and alsochanges the triglyceride composition, resulting in the formation ofcompounds that have a higher melting point than those found in commonsoybean oil. The melting point of high stearate soybean oil is belowroom temperature, but solid fats are present that crystallize when theoil is stored at room temperature.

Example 5 Stability of High Stearate Soybean Oil

TABLE 6 Stability assays of soybean oil Common soybean High stearatesoybean Stability criteria oil oil Inherent stability 7.0 3.9 Calculatediodine value 132 101 Active oxygen method 8-10 40

Inherent stability is a calculated relative reactivity with oxygen, withhigher values denoting a greater predisposition to oxidation (M.Erickson and N. Frey, Food Technology, 50: 63-68 (1994)). Iodine valuesrepresent calculated reactivities with elemental iodine, with highervalues indicating greater reactivities (Official and Tentative Methods,American Oil Chemists' Society, Cd 1b-87, Champaign, Ill.). The activeoxygen method assay simulates thermal breakdown encountered duringcooking, with higher values representing greater thermal stability(Official and Tentative Methods, American Oil Chemists' Society, Cd12-57(93), Champaign, Ill.). The increase in saturated andmonounsaturated fatty acids, and the decrease in polyunsaturated fattyacids in high stearate soybean oil results in a greater thermalstability when compared to common soybean oil. The greater thermalstability of high stearate soybean oil when compared to common soybeanoil agrees with predictions based upon the calculated inherentstabilities and iodine values.

Example 6 Solids Profile of Soybean Oils

TABLE 7 Solids present at various temperatures Solids present attemperature Oil type 50° F. 70° F. 80° F. 92° F. 104° F. Common soybeanoil — — — — — High stearate (H4152) 9.8 — — — — Interesterified high 7.03.5 3.0 2.0 0.7 stearate

Solids were evaluated by the solids fat index using dilatometry. Valuesrepresent percent solids in a sample at the given temperature.Interesterified high stearate oil was prepared byinteresterificationaccording to the method of Erickson (Practical Handbook of SoybeanProcessing and Utilization, American Oil Chemists' Society Press,Champaign Ill., 1995). In contrast, soybean oil A6 having high stearic,normal levels of palmitic and linolenic, and low levels of oleic andlinoleic acids, has no solids present at 24.7° C. and highertemperatures.

Example 7 Application of High Stearate Soybeans in Soy Based Foods

A. To prepare tofu and soymilk, 50 grams of high stearate soybeans weresoaked in 150 grams water overnight and drained. Soybeans were rinsedwith water. 185 grams of boiling water was added, and the mixturepureed. 315 grams of water were added, and the mixture heated to 100° C.for 10 minutes. Okara (filtered solids) was extracted using the JuicemanJunior machine (Salton Maxim, Mt. Prospect, Ill.) to dehull the beans.The hot soymilk should be approximately 8% solids. For firm tofu, 1.5grams glucono-δ-lactone (Aldrich, Milwaukee, Wis.) was added. The liquidwas allowed to coagulate for 15 to 20 minutes at 90° C. with lightstirring. The liquid was allowed to cool and form tofu.

B. To prepare soymilk, 150 grams of high stearate soybeans were soakedin 500 mL water overnight and drained. Soaked soybeans were rinsed withwater. The beans were equally divided into two portions. Each portionwas ground with 400 mL water in an Oster blender (Sunbeam, Delray Beach,Fla.) at the highest speed for 1.5 minutes. The combined slurry from thetwo portions was manually filtered through cloth. The solid residue wasdiscarded. The filtrate was heated to 95° C. for 10 minutes to preparesoymilk. To prepare tofu, the hot soymilk was cooled to about 75° C.,and 5 grams of either calcium sulfate or glucono-δ-lactone was added.The mixture was allowed to stand for 30 minutes to form curd, whichbecame silken tofu. To prepare firm tofu, the hot curd was broken,placed in a mold, and pressed to release the whey.

Tofu prepared from high stearate oil soybeans had a consistency that wasfirmer and more creamy than tofu prepared from standard controlsoybeans.

Example 8 Applications of Full Fat Soy Flour in Baked Foods

High stearate soybeans were processed into full fat soy flour using thestandard industry protocol (Practical Handbook of Soybean Processing andUtilization, American Oil Chemists' Society Press, Champaign Ill.,1995). The flour can be added to baked products at high levels (15-20%)to increase the protein content without affecting the texture of thebaked products. Flour obtained from high stearate soybeans can be usedin an array of food products including candies, gravies, sauces, frozendesserts, pastas, meat products, and baked goods.

Example 9 Margarine Formulation Using High Stearate Soybean Oil

TABLE 8 Margarine composition Ingredient Weight percent Water 16.85 Wheyprotein 0.4 Salt 1.9 Lecithin 0.4 Monoglyceride (Super G7, A C Humko Co)0.45 Sodium benzoate 0.001 Interesterified high stearate oil 80.0Flavor, color to 100% (I-1435, Fries & Fries)

The oil was heated to 65° C. in a microwave oven. Flavors were withheldand added to the oil phase immediately before the oil phase was combinedwith the remaining components which had been heated to 50° C. in amicrowave oven. The two phases were combined and mixed for 20 minutes at2000 rpm in a Dispermat unit (VMA-Getzmann, Germany) maintained at 60°C. The margarine was filled into one pound tubs and placed at 40° C. forcrystallization. The margarine product was easily spreadable whenremoved from refrigeration after one day, and after long term storage offour weeks. The margarine exhibited good room temperature stability aswell as excellent flavor release and structure.

Example 10 Formulation of all-Purpose Shortenings with InteresterifiedHigh Stearate Oil

Commercial shortenings, such as CRISCO (Proctor and Gamble, Cincinnati,Ohio), are composed of a hydrogenated soybean oil combined with a fullyhydrogenated cottonseed oil component and an emulsifier such as amonoglyceride. The soybean oil basestocks generally have a trans fattyacid content of greater than 15% (w/w), and frequently greater than 25%(w/w). The interesterified high stearate soybean oil has sufficientsolids such that when combined with the fully hydrogenated cottonseedoil and monoglyceride, it gives a texture and consistency similar to theCRISCO product upon votation (rapid chilling and working of fat, Weiss,T. J., Food oils and Their Uses, Avi Publishing Co., Westport, Conn.,1983) and crystallization. Nitrogen is added to the formulation tomodify the final density and solidity of the product. A 15% overrun ofgas corresponds to a 15% reduction in density of the shortening incomparison to the density before addition of nitrogen.

TABLE 9 Shortening formulation Component Percent by mass Interesterifiedhigh stearate oil 89.2 Cottonseed oil (5 iodine value max.) 7.8Monoglyceride (Super G7, A C Humko Co) 3.0 Nitrogen gas 15%

Example 11 Shelf Life Testing

Non-hydrogenated oils are preferred over partially hydrogenated oils dueto costs and improved acceptance by the consumer. However, the shortshelf stability of non-hydrogenated oils severely limit their foodapplications. The interesterified high stearate soybean oil was used ina potato chip rancidity assay to determine the applicability in thepreparation of fried foods.

TABLE 10 Schaal oven test Oil Days to detect rancid odor Common soybeanoil 2-3 All-purpose shortening (CRISCO) 6-7 Interesterified highstearate oil 11 (all purpose shortening formulation

The rancidity assay was performed using a Schaal oven test at 62° C.(Warner, K. and Eskin, N. M., Methods to Assess Quality and Stability ofOils and Fat-Containing Foods, American Oil Chemists' Society Press,Champaign, Ill., 1995). Interesterified high stearate oil demonstrated amarked increase in stability, as indicated by the longer duration oftime required to detect an undesirable odor.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1-53. (canceled)
 54. Soybean seed oil comprising greater than about 20%stearate and less than about 53% stearate.
 55. The soybean seed oil ofclaim 54, further comprising about 50 weight percent or greater stearateexpressed as a component of total fatty acids contained in said oil. 56.The soybean seed oil of claim 54, further comprising about 25 percent orgreater of said total fatty acids in a saturated-unsaturated-saturatedform.
 57. The soybean seed oil of claim 54, further comprising about 25percent or greater stearate as the fatty acid found at the sn-1 and sn-3positions of seed triglycerides.
 58. The soybean seed oil of claim 57,further comprising oleic acid as the predominant fatty acid at the sn-2position of seed triglycerides.
 59. The soybean seed oil of claim 54,wherein said oil without chemical modification comprises about 24% orgreater stearate and less than 3% linoleate.
 60. The soybean seed oil ofclaim 54, wherein said oil without chemical modification has a meltingpoint below room temperature.
 61. A food product comprising the soybeanseed oil of claim 54, wherein said food product is selected from thegroup consisting of salad oil, cooking oil, margarine, shortening,soymilk, tofu, and soy flour.
 62. A method of producing a soybean seedhaving above 15% stearate comprising the steps of: (a) growing anoilseed crop having inserted into its genome an exogenous DNA encoding aplant FatA thioesterase; and (b) harvesting said oilseed crop, whereinthe seed of said oilseed crop comprises an endogenous oil comprisinggreater than 15% stearate.